Rat cauda epidiidymal (CE) sperm are stored immotile in the epididymis and become vigorously motile when ther are ejaculated. We found that these sperm are mechanically immobilized in the cauda epididymis by "immobilin", a large mucin-like glycoprotein that makes the epedidymal fluid highly viscoelastic. During ejaculation, immobilin is both diluted and degraded, greatly reducing the biscoelasticity of the fluid and thereby initiating sperm motility. In this grant application we propse to continue our research on the initiation of sperm motility. Our major aims are: 1) To fully characterize the rheological properties of CE fluid using an oscillating ball microrheometer. 2) To carry out a thorough biochemical analysis of immobilin, including the characterization of its carbohydrate side chains and the disulfide bonds and other crosslinks that help to produce high viscoelasticity. 3) To determine the cellular changes that occur in a sperm cell as it first begins to swim by using selective extracellular electrodes to measure changes in sperm cell properties such an membrane potential, cytoplasmic calcium activity, and respiration rate, and by using other investigate the "probing" mechanism by which a sperm cell detects the change in external viscoelasicity that allows it to initiate motility. For example, we will analyze motility initiation using high speed and high resolution video tapes to determine how the sperm probes its enviroment, and how the probing mechanism responds to different rheological properties of the surrounding fluid. 5) Finally, we will investigate how the mechanisms of motility initiation in other animals compares to the mechanism in rat. Our motivation for pursuing this research is to contribute to the development of better baarrier methods of contraception.